The OVERALL OBJECTIVE of this proposal is to define the cellular mechanisms culminating in lethal injury of cholangiocytes and hepatocytes in models relevant to human cholestatic liver diseases (e.g., primary sclerosing cholangitis). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) contributes to both cholangiocyte and hepatocyte injury in models relevant to these liver diseases. Thus, our long term objectives are to understand the cellular and subcellular mechanisms causing cholangiocyte and hepatocyte injury by this death ligand. Our distinctive preliminary data implicate phosphofurin acidic cluster sorting-2 (PACS-2) protein as a critical regulator of TRAIL-induced cytotoxicity by a lysosomal cell death pathway. Cellular inhibitor of apoptosis-1 (cIAP-1) regulates the lysosomal cell death pathway by controlling PACS-2 protein levels. Cholangiocytes appear to be intrinsically sensitive to TRAIL killing via this pathway in vivo, whereas hepatocytes in vivo only become sensitive to TRAIL toxicity during cholestasis. Based upon this preliminary data, we propose the novel CENTRAL HYPOTHESIS that TRAIL induces liver cell death by a lysosomal pathway dependent upon PACS-2, and cholangiocytes, which express less cIAP-1 than hepatocytes, are preferentially sensitive to this pathway in vivo. We will now employ current and complementary molecular, biochemical and cell biological approaches to ascertain how TRAIL triggers this organelle-based pathway of apoptosis. Our proposal has three SPECIFIC AIMS. FIRST, we will directly test the hypothesis that PACS-2 mediates lysosomal membrane permeabilization during TRAIL cytotoxicity by: a) binding Bim, a BH3-only protein, which recruits and activates Bax, a potent proapoptotic protein of the Bcl-2 family; and b) facilitating Bax oligomerization within lysosomal membranes which causes lysosomal disruption. SECOND, we will directly test the hypothesis that cIAP-1, an E3 ligase, modulates TRAIL cytotoxicity by: a) regulating PACS- 2 protein levels via an ubiquitination-dependent mechanism resulting in its proteasomal degradation; and b) by regulating the lysosomal cell death pathway through control of PACS-2 protein levels. FINALLY, we will directly test the hypothesis that: PACS-2 deletion is salutary in models relevant to cholestatic liver injury by: a) reducing TRAIL-mediated cholangiocyte injury in vivo in a model reminiscent of primary sclerosing cholangitis; and b) by reducing hepatocyte injury in vivo during obstructive cholestasis in the bile duct ligated mouse. The proposal is innovative as it tests new concepts for TRAIL cytotoxicity using sophisticated technologies. The information generated will provide a framework for the development of novel therapeutic strategies effective for attenuating TRAIL-mediated human liver injury during cholestasis.